Flexural rigidity of individual microtubules measured by a buckling force with optical traps

We used direct buckling force measurements with optical traps to determine the flexural rigidity of individual microtubules bound to polystyrene beads. To optimize the accuracy of the measurement, we used two optical traps and antibody-coated beads to manipulate each microtubule. We then applied a new analytical model assuming nonaxial buckling. Paclitaxel-stabilized microtubules were polymerized from purified tubulin, and the average microtubule rigidity was calculated as 2.0 x 10(-24) Nm2 using this novel microtubule buckling system. This value was not dependent on microtubule length. We also measured the rigidity of paclitaxel-free microtubules, and obtained the value of 7.9 x 10(-24) Nm2, which is nearly four times that measured for paclitaxel-stabilized microtubules.     

Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape

Microtubules are long, proteinaceous filaments that perform structural functions in eukaryotic cells by defining cellular shape and serving as tracks for intracellular motor proteins. We report the first accurate measurements of the flexural rigidity of microtubules. By analyzing the thermally driven fluctuations in their shape, we estimated the mean flexural rigidity of taxol-stabilized microtubules to be 2.2 x 10(-23) Nm2 (with 6.4% uncertainty) for seven unlabeled microtubules and 2.1 x 10(-23) Nm2 (with 4.7% uncertainty) for eight rhodamine-labeled microtubules. These values are similar to earlier, less precise estimates of microtubule bending stiffness obtained by modeling flagellar motion. A similar analysis on seven rhodamine-phalloidin- labeled actin filaments gave a flexural rigidity of 7.3 x 10(-26) Nm2 (with 6% uncertainty), consistent with previously reported results. The flexural rigidity of these microtubules corresponds to a persistence length of 5,200 microns showing that a microtubule is rigid over cellular dimensions. By contrast, the persistence length of an actin filament is only approximately 17.7 microns, perhaps explaining why actin filaments within cells are usually cross-linked into bundles. The greater flexural rigidity of a microtubule compared to an actin filament mainly derives from the former's larger cross-section. If tubulin were homogeneous and isotropic, then the microtubule's Young's modulus would be approximately 1.2 GPa, similar to Plexiglas and rigid plastics. Microtubules are expected to be almost inextensible: the compliance of cells is due primarily to filament bending or sliding between filaments rather than the stretching of the filaments themselves.     

Mechanics of microtubule bundles in pillar cells from the inner ear.

The mechanical properties of cross-linked microtubule bundles were measured from outer pillar cells isolated from the mammalian inner ear. Measurements were made using a three-point bending test and were incorporated into a mathematical model designed to distinguish between the stiffness contributions from microtubules and their cross-linking proteins. Outer pillar cells were composed of 1000-3000 parallel bundled microtubules in a square array that was interdigitated and cross-linked with actin filaments. The average midpoint bending stiffness of intact cells was 7 x 10(-4) N/m. After removal of both the actin filaments and cross-links with detergent in the presence of DNase I, the square array was disrupted and the stiffness decreased by a factor of 4, to 1.7 x 10(-4) N/m. The bending modulus for individual microtubules was calculated to be 7 x 10(-23) Nm2, and the Young's modulus for these 15 protofilament microtubules was 2 x 10(9) Pa. The shear modulus between microtubules in intact cells was calculated to be 10(3) Pa. It was concluded that cross-linking proteins provided shear resistance between microtubules, which resulted in a fourfold increase in stiffness. The model can be used to estimate the mechanical properties of cross-linked microtubule bundles in cells from which direct measurements are not available.     

Dynamics of microtubule depolymerization in monocytes

Human monocytes, which contain few interphase microtubules (35.+/- 7.7), were used to study the dynamics of microtubule depolymerization. Steady-state microtubule assembly was abruptly blocked with either high concentrations of nocodazole (10 micrograms/ml) or exposure to cold temperature (3 degrees C). At various times after inhibition of assembly, cells were processed for anti-tubulin immunofluorescence microscopy. Stained cells were observed with an intensified video camera attached to the fluorescence microscope. A tracing of the entire length of each individual microtubule was made from the image on the television monitor by focusing up and down through the cell. The tracings were then digitized into a computer. All microtubules were seen to originate from the centrosome, with an average length in control cells of 7.1 +/- 2.7 microns (n = 957 microtubules). During depolymerization, the total microtubule polymer and the number of microtubules per cell decreased rapidly. In contrast, there was a slow decrease in the average length of the persisting microtubules. The half-time for both the loss of total microtubule polymer and microtubule number per cell was approximately 40 s for nocodazole-treated cells. The rate-limiting step in the depolymerization process was the rate of initiation of disassembly. Once initiated, depolymerization appeared catastrophic. Further kinetic analysis revealed two classes of microtubules: 70% of the microtubule population was very labile and initiated depolymerization at a rate approximately 23 times faster than a minor population of persistent microtubules. Cold treatment yielded qualitatively similar characteristics of depolymerization, but the initiation rates were slower. In both cases there was a significant asynchrony and heterogeneity in the initiation of depolymerization among the population of microtubules.     

Molecular recognition of taxol by microtubules. Kinetics and thermodynamics of binding of fluorescent taxol derivatives to an exposed site

We have determined the kinetic scheme and the reaction rates of binding to microtubules of two fluorescent taxoids, 7-O-[N-(4'-fluoresceincarbonyl)-l-alanyl]Taxol (Flutax-1) and 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-l-alanyl]Taxol (Flutax-2). Flutax-1 and Flutax-2 bind to microtubules with high affinity (K(a) approximately 10(7) m(-1), 37 degrees C). The binding mechanism consists of a fast bimolecular reaction followed by at least two monomolecular rearrangements, which were characterized with stopped-flow techniques. The kinetic constants of the bimolecular reaction were 6.10 +/- 0.22 x 10(5) m(-1) s(-1) and 13.8 +/- 1.8 x 10(5) m(-1) s(-1) at 37 degrees C, respectively. A second slow binding step has been measured employing the change of fluorescence anisotropy of the probe. The reversal of this reaction is the rate-limiting step of dissociation. A third step has been detected using small angle x-ray scattering and involves a 2-nm increase in the diameter of microtubules. It is suggested that the first step entails the binding of the Taxol moiety and the second a relative immobilization of the fluorescent probe. The equilibrium and some kinetic measurements required the use of stabilized cross-linked microtubules, which preserved taxoid binding. The results indicate that the Taxol binding site is directly accessible, in contrast with its location at lumen in the current model of microtubules. An alternative structural model is considered in which the binding site is located between protofilaments, accessible from the microtubule surface.     

Interaction of tubulin and cellular microtubules with the new antitumor drug MDL 27048. A powerful and reversible microtubule inhibitor

We have characterized the binding of trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2- propen- 1-one (MDL 27048) to purified procine brain tubulin, and the inhibition of microtubule assembly by this compound in vitro and using cultured cells. Binding measurements were performed by difference absorption and fluorescence spectroscopy. MDL 27048 binds to one site/tubulin heterodimer with an apparent equilibrium constant Kb = (2.8 +/- 0.8) X 10(6) M-1 (50 mM 2-(N-morpholino)ethanesulfonic acid, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 0.5 mM MgCl2, 0.1 mM GTP buffer, pH 6.7, at 25 degrees C). Podophyllotoxin displaced the binding of MDL 27048, suggesting an overlap with the colchicine-binding site. Assembly of purified tubulin into microtubules was inhibited by substoichiometric concentrations of MDL 27048, which also induced a slow depolymerization of preassembled microtubules. The cytoplasmic microtubules of PtK2 cells were disrupted in a concentration and time-dependent manner by MDL 27048, as observed by indirect immunofluorescence microscopy. Maximal depolymerization took place with 2 X 10(-6) M MDL 27048 in 3 h. When the inhibitor was washed off from the cells, fast microtubule assembly (approximately 8 min) and complete reorganization of the cytoplasmic microtubule network (15-30 min) were observed. MDL 27048 also induced mitotic arrest in SV40-3T3 cell cultures. Due to all these properties, this anti-tumor drug constitutes a new and potent microtubule inhibitor, characterized by its specificity and reversibility.     

Fast kinetics of Taxol binding to microtubules. Effects of solution variables and microtubule-associated proteins

The kinetics of Taxol association to and dissociation from stabilized microtubules has been measured by competition with the reference fluorescent derivative Flutax-1 (Diaz, J. F., Strobe, R., Engelborghs, Y., Souto, A. A., and Andreu, J. M. (2000) J. Biol. Chem. 275, 26265-26276). The association rate constant at 37 degrees C is k(+) = (3.6 +/- 0.1) x 10(6) m(-1) s(-1). The reaction profile is similar to that of the first step of Flutax-1 binding, which probably corresponds to the binding of the Taxol moiety. The rate constant of the initial binding of Flutax-1 is inversely proportional to the viscosity of the solution, which is compatible with a diffusion-controlled reaction. Microtubule-associated proteins bound to the microtubule outer surface slow down the binding of Flutax-1 and Flutax-2 10-fold. The binding site is fully accessible to Flutax-2 in native cytoskeletons of PtK2 cells; the observed kinetic rates of Flutax-2 microtubule staining and de-staining are similar to the reaction rates with microtubule associated proteins-containing microtubules. The kinetic data prove that taxoids bind directly from the bulk solution to an exposed microtubule site. Several hypotheses have been analyzed to potentially reconcile these data with the location of a Taxol-binding site at the model microtubule lumen, including dynamic opening of the microtubule wall and transport from an initial Taxol-binding site at the microtubule pores.     

Microtubule depolymerization promotes particle and chromosome movement in vitro

We have developed a system for studying the motions of cellular objects attached to depolymerizing microtubules in vitro. Radial arrays of microtubules were grown from lysed and extracted Tetrahymena cells attached to a glass coverslip that formed the top of a light microscope perfusion chamber. A preparation of chromosomes, which also contained vesicles, was then perfused into the chamber and allowed to bind to the microtubule array. The concentration of tubulin was then reduced by perfusing buffer that lacked both tubulin and nucleotide triphosphates, and the resulting microtubule depolymerization was observed by light microscopy. A fraction of the bound objects detached in the flow and washed away, while others stabilized the microtubules to which they were bound. Some of the particles and chromosomes, however, moved in toward the Tetrahymena ghost as their associated microtubules shortened. The mean speeds for particles and chromosomes were 26 +/- 20 and 15 +/- 12 microns/min, respectively. These motions occurred when nucleotide triphosphate levels were very low, as a result of either dilution or by the action of apyrase. Furthermore, the motions were unaffected by 100 microM sodium orthovanadate, suggesting that these forces are not the result of ATP hydrolysis by a minus end-directed mechanoenzyme. We conclude that microtubule depolymerization provided the free energy for the motions observed. All the objects that we studied in detail moved against a stream of buffer flowing at approximately 100 microns/s, so that the force being developed was at least 10(-7) dynes. This force is large enough to contribute to some forms of motility in living cells.     

A bending mode analysis for growing microtubules: evidence for a velocity-dependent rigidity

Microtubules are dynamic protein polymers that continuously switch between elongation and rapid shrinkage. They have an exceptional bending stiffness that contributes significantly to the mechanical properties of eukaryotic cells. Measurements of the persistence length of microtubules have been published since 10 years but the reported values vary over an order of magnitude without an available explanation. To precisely measure the rigidity of microtubules in their native growing state, we adapted a previously developed bending mode analysis of thermally driven shape fluctuations to the case of an elongating filament that is clamped at one end. Microtubule shapes were quantified using automated image processing, allowing for the characterization of up to five bending modes. When taken together with three other less precise measurements, our rigidity data suggest that fast-growing microtubules are less stiff than slow-growing microtubules. This would imply that care should be taken in interpreting rigidity measurements on stabilized microtubules whose growth history is not known. In addition, time analysis of bending modes showed that higher order modes relax more slowly than expected from simple hydrodynamics, possibly by the effects of internal friction within the microtubule.     

Role of microtubules in LPS-induced macrophage inflammatory protein-2 production from rat pneumocytes

We have recently demonstrated that primary cultured rat pneumocytes produce macrophage inflammatory protein-2 (MIP-2) in response to lipopolysaccharide (LPS) stimulation. In this study, we found that brefeldin A, by blocking anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus, decreased LPS-induced MIP-2 in the culture medium and increased its storage in cells. This suggests that MIP-2 is secreted via a pathway from the ER to the Golgi apparatus, a process commonly regulated by microtubules. We further found that LPS induced depolymerization of microtubules as early as 1 min after LPS stimulation, and it lasted at least for 4 h. Preventing depolymerization of microtubules with paclitaxel (Taxol; 10 nM to 10 microM) partially inhibited LPS-induced MIP-2 production, whereas the microtubule-depolymerizing agents colchicine (1-10 microM) and nocodazole (1-100 microM) increased LPS-induced MIP-2 protein production without affecting MIP-2 mRNA expression. These results suggest that in pneumocytes, LPS-induced microtubule depolymerization is involved in LPS-induced MIP-2 production and that secretion of MIP-2 from pneumocytes is via the ER-Golgi pathway.     

Domains of Neuronal Microtubule-associated Proteins and Flexural Rigidity of Microtubules

Microtubules are flexible polymers whose mechanical properties are an important factor in the determination of cell architecture and function. It has been proposed that the two most prominent neuronal microtubule-associated proteins (MAPs), tau and MAP2, whose microtubule binding regions are largely homologous, make an important contribution to the formation and maintenance of neuronal processes, putatively by increasing the rigidity of microtubules. Using optical tweezers to manipulate single microtubules, we have measured their flexural rigidity in the presence of various constructs of tau and MAP2c. The results show a three- or fourfold increase of microtubule rigidity in the presence of wild-type tau or MAP2c, respectively. Unexpectedly, even low concentrations of MAPs promote a substantial increase in microtubule rigidity. Thus at ~20% saturation with full-length tau, a microtubule exhibits >80% of the rigidity observed at near saturating concentrations. Several different constructs of tau or MAP2 were used to determine the relative contribution of certain subdomains in the microtubule-binding region. All constructs tested increase microtubule rigidity, albeit to different extents. Thus, the repeat domains alone increase microtubule rigidity only marginally, whereas the domains flanking the repeats make a significant contribution. Overall, there is an excellent correlation between the strength of binding of a MAP construct to microtubules (as represented by its dissociation constant K_d) and the increase in microtubule rigidity. These findings demonstrate that neuronal MAPs as well as constructs derived from them increase microtubule rigidity, and that the changes in rigidity observed with different constructs correlate well with other biochemical and physiological parameters.     

Effect of microtubule stabilization on the freezing tolerance of mesophyll cells of spinach

Freezing, dehydration, and supercooling cause microtubules in mesophyll cells of spinach (Spinacia oleracea L. cv Bloomsdale) to depolymerize (ME Bartolo, JV Carter, Plant Physiol [1991] 97: 175-181). The objective of this study was to determine whether the LT₅₀ (lethal temperature: the freezing temperature at which 50% of the tissue is killed) of spinach leaf tissue can be changed by diminishing the extent of microtubule depolymerization in response to freezing. Also examined was how tolerance to the components of extracellular freezing, low temperature and dehydration, is affected by microtubule stabilization. Leaf sections of nonacclimated and cold-acclimated spinach were treated with 20 micromolar taxol, a microtubule-stabilizing compound, prior to freezing, supercooling, or dehydration. Taxol stabilized microtubules against depolymerization in cells subjected to these stresses. When pretreated with taxol both nonacclimated and cold-acclimated cells exhibited increased injury during freezing and dehydration. In contrast, supercooling did not injure cells with taxol-stabilized microtubules. Electrolyte leakage, visual appearance of the cells, or a microtubule repolymerization assay were used to assess injury. As leaves were cold-acclimated beyond the normal period of 2 weeks taxol had less of an effect on cell survival during freezing. In leaves acclimated for up to 2 weeks, stabilizing microtubules with taxol resulted in death at a higher freezing temperature. At certain stages of cold acclimation, it appears that if microtubule depolymerization does not occur during a freeze-thaw cycle the plant cell will be killed at a higher temperature than if microtubule depolymerization proceeds normally. An alternative explanation of these results is that taxol may generate abnormal microtubules, and connections between microtubules and the plasma membrane, such that normal cellular responses to freeze-induced dehydration and subsequent rehydration are blocked, with resultant enhanced freezing injury.     

Vibration and length-dependent flexural rigidity of protein microtubules using higher order shear deformation theory

Microtubules are hollow cylindrical filaments of the eukaryotic cytoskeleton characterized by extremely low shear modulus. A remarkable controversy has occurred in the literature, regarding the length dependence of flexural rigidity of microtubules predicted by the classical elastic beam model. In this study, a higher order shear deformable beam model for microtubules is employed to study unexplained length-dependent flexural rigidity and Young’s modulus of microtubules reported in the literature. The formulation allows for warping of the cross-section of the microtubule and eliminates the need for using arbitrary shear correction coefficients as in other theories. It is showed that vibration frequencies predicted by the present parabolic shear deformation theory (PSDT) are much lower than that given by the approximate isotropic beam model for shorter microtubules, although the two models give almost identical results for sufficiently long microtubules. It is confirmed that transverse shearing and the warping of the cross-section of microtubules are mainly responsible for the length-dependent flexural rigidity of an isolated microtubule reported in the literature, which cannot be explained by the widely used Euler-Bernoulli beam model. Indeed, the length-dependent flexural rigidity predicted by the present model is found to be in qualitative agreement with the existing experimental data ( [Kurachi et al., 1995] and [Pampaloni et al., 2006]). These results recommend that the parabolic shear deformation-beam theory offers a unified simple 1D model, which can capture the length dependence of flexural rigidity and be applied to various static and dynamic problems of microtubule mechanics.     

The minus end-directed motor Kar3 is required for coupling dynamic microtubule plus ends to the cortical shmoo tip in budding yeast

The budding yeast shmoo tip is a model system for analyzing mechanisms coupling force production to microtubule plus-end polymerization/depolymerization. Dynamic plus ends of astral microtubules interact with the shmoo tip in mating yeast cells, positioning nuclei for karyogamy. We have used live-cell imaging of GFP fusions to identify proteins that couple dynamic microtubule plus ends to the shmoo tip. We find that Kar3p, a minus end-directed kinesin motor protein, is required, whereas the other cytoplasmic motors, dynein and the kinesins Kip2p and Kip3p, are not. In the absence of Kar3p, attached microtubule plus ends released from the shmoo tip when they switched to depolymerization. Furthermore, microtubules in cells expressing kar3-1, a mutant that results in rigor binding to microtubules [2], were stabilized specifically at shmoo tips. Imaging of Kar3p-GFP during mating revealed that fluorescence at the shmoo tip increased during periods of microtubule depolymerization. These data are the first to localize the activity of a minus end-directed kinesin at the plus ends of microtubules. We propose a model in which Kar3p couples depolymerizing microtubule plus ends to the cell cortex and the Bim1p-Kar9p protein complex maintains attachment during microtubule polymerization. In support of this model, analysis of Bim1p-GFP at the shmoo tip results in a localization pattern complementary to that of Kar3p-GFP.     

Base plate mechanics of the barnacle Balanus amphitrite (=Amphibalanus amphitrite)

The mechanical properties of barnacle base plates were measured using a punch test apparatus, with the purpose of examining the effect that the base plate flexural rigidity may have on adhesion mechanics. Base plate compliance was measured for 43 Balanus amphitrite (=Amphibalanus amphitrite) barnacles. Compliance measurements were used to determine flexural rigidity (assuming a fixed-edge circular plate approximation) and composite modulus of the base plates. The barnacles were categorized by age and cement type (hard or gummy) for statistical analyses. Barnacles that were 'hard' (> or =70% of the base plate thin, rigid cement) and 'gummy' (>30% of the base plate covered in compliant, tacky cement) showed statistically different composite moduli but did not show a difference in base plate flexural rigidity. The average flexural rigidity for all barnacles was 0.0020 Nm (SEM +/- 0.0003). Flexural rigidity and composite modulus did not differ significantly between 3-month and 14-month-old barnacles. The relatively low flexural rigidity measured for barnacles suggests that a rigid punch approximation is not sufficient to account for the contributions to adhesion mechanics due to flexing of real barnacles during release.     

XKCM1 acts on a single protofilament and requires the C terminus of tubulin

The stability of microtubules during the cell-cycle is regulated by a number of cellular factors, some of which stabilize microtubules and others that promote breakdown. XKCM1 is a kinesin-like protein that induces microtubule depolymerization and is required for mitotic spindle assembly. We have examined the binding and depolymerization effects of XKCM1 on different tubulin polymers in order to learn about its mechanism of action. Zinc-induced tubulin polymers, characterized by an anti-parallel protofilament arrangement, are depolymerized by XKCM1, indicating that this enzyme acts on a single protofilament. GDP-tubulin rings, which correspond to the low-energy state of tubulin, are stable only under conditions that inhibit XKCM1 depolymerizing activity, but can be stabilized by XKCM1 bound to AMPPNP. Tubulin polymers made of subtilisin-treated tubulin (lacking the tubulin C-terminal tail) are resistant to XKCM1-induced depolymerization, suggesting that the interaction of the acidic tail of tubulin with basic residues in XKCM1 unique to Kin I proteins is required for depolymerization.     

Temperature dependence of the flexural rigidity of single microtubules

Although the flexural rigidity of a microtubule has previously been estimated by various methods, its temperature dependence has never been systematically examined. Here, we measured the flexural rigidity of a single taxol-stabilized microtubule from thermal fluctuation of the free end of a microtubule, the other end of which was fixed, at different temperatures; the results showed that the flexural rigidity is 2.54 × 10⁻²⁴ N m² independent of temperature in the range of 20-35 °C. Next, we applied temperature pulse microscopy (TPM) [K. Kawaguchi, S. Ishiwata, Thermal activation of single kinesin molecules with temperature pulse microscopy. Cell Motil. Cytoskeleton 49 (2001) 41-47; H. Kato, T. Nishizaka, T. Iga, K. Kinosita Jr., S. Ishiwata, Imaging of thermal activation of actomyosin motors. Proc. Natl. Acad. Sci. USA 96 (1999) 9602-9606], which created the temperature gradient (1-2 °C/μm) along a microtubule gliding on kinesins in the presence of ATP. As a result, the gliding microtubule was buckled between two interacting kinesin molecules, when the microtubule had been propelled faster by the rear kinesin (higher temperature) and slower by the front one (lower temperature). By estimating the critical force to induce buckling of a microtubule, the flexural rigidity of a microtubule was estimated to be (2.7-7.8) × 10⁻²⁴ N m², which was in good agreement with the value determined above. We discuss the buckling process based on the temperature dependence of the force-velocity relationship of kinesin motility.     

Eg5 causes elongation of meiotic spindles when flux-associated microtubule depolymerization is blocked

In higher eukaryotes, microtubules (MT) in both halves of the mitotic spindle translocate continuously away from the midzone in a phenomenon called poleward microtubule flux. Because the spindle maintains constant length and microtubule density, this microtubule translocation must somehow be coupled to net MT depolymerization at spindle poles. The molecular mechanisms underlying both flux-associated translocation and flux-associated depolymerization are not well understood, but it can be predicted that blocking pole-based destabilization will increase spindle length, an idea that has not been tested in meiotic spindles. Here, we show that simultaneous addition of two pole-disrupting reagents p50/dynamitin and a truncated version of Xklp2 results in continuous spindle elongation in Xenopus egg extracts, and we quantitatively correlate this elongation rate with the poleward translocation of stabilized microtubules. We further use this system to demonstrate that this poleward translocation requires the activity of the kinesin-related protein Eg5. These results suggest that Eg5 is responsible for flux-associated MT translocation and that dynein and Xklp2 regulate flux-associated microtubule depolymerization at spindle poles.     

Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP.

The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta--gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 x 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.     

The stabilization of microtubules in isolated spindles by tubulin-colchicine complex

We have analyzed the effect of colchicine and tubulin dimer-colchicine complex (T-C) on microtubule assembly in mitotic spindles. Cold- and calcium-labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly-disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a half-time of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 microM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 microM T-C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T-C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T-C in vivo. These results support the current view that colchicine does not directly promote microtubule depolymerization. Rather, it is T-C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation. In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly.(ABSTRACT TRUNCATED AT 250 WORDS)